The healthy intestines of mammals contain billions of colonies of bacteria, bacteria that have evolved with mammals that typically, and under optimal health conditions of the mammal, function in a synergistic manner with the host mammal. Unfortunately, the widespread use of antibiotics, especially broad-spectrum antibiotics, has led to deleterious pathological conditions. The physiological health of the gastrointestinal tract is dependent on the health of its bacterial population, or its “microflora.” A plurality of individual bacterial species inhabit the gastrointestinal tract and their growth and metabolism depend primarily upon the substrates available to them, most of which are derived from the diet. The administration of antibiotics does kill harmful pathogens, but antibiotics do not discriminate between harmful pathogens and beneficial, non-pathogenic microorganisms resident in the flora. The beneficial microorganisms, typically the lactic acid producing microorganisms, are destroyed by antibiotics and impair health and digestive function. A result of the proliferation of harmful bacteria species is infection from toxic by-products resulting in diarrhea and damage to mucosal lining. Absorption and assimilation of food and bioactive ingredients are disturbed and at the same time there may be relapse (the return of infections and their accompanying signs and symptoms).
Other deleterious results of indiscriminate use of antibiotics is the generation of multiple antibiotic-resistant pathogens and occurrence of secondary opportunistic infections which often result from the depletion of lactic acid producing and other beneficial flora within the gastrointestinal tract. Methicillin-resistant Staphylococcus aureus (MRSA) infections and vancomycin-resistant Enterococci (VRE) have been reported. The development of such resistance has led to numerous reports of systemic infections that are not treatable with conventional antibiotic therapies.
Clostridium difficile is a Gram-positive facultative anaerobic bacteria that is widespread in human intestinal flora that, when its growth is unchecked by other bacteria, produces toxins that cause debilitating and life threatening colitis. Clostridium difficile, often called C. difficile or “C. diff,” can cause symptoms ranging from diarrhea to life-threatening inflammation of the colon. Illness from C. difficile most commonly affects older adults in hospitals or in long term care facilities and typically occurs after use of antibiotic medications. In recent years, C. difficile infections have become more frequent, more severe and more difficult to treat. Each year, tens of thousands of people in the United States became ill from C. difficile, including some otherwise healthy people who are not hospitalized or taking antibiotics.
C. difficile bacteria can be found throughout the environment—in soil, air, water, and human and animal feces. A small number of healthy people naturally carry the bacteria in their large intestine. C. difficile is most commonly found in hospitals, nursing homes, extended care facilities, nurseries for newborn infants, institutions and other health care facilities, where a much higher percentage of people carry the bacteria. C. difficile may also be present in communal animal environments such as kennels, barns, and stables. When C. difficile grows unchecked, it produces two primary toxins, known as Toxin A and Toxin B, that trigger the formation of a psuedomembrane in the colon line that interferes with the normal function of the organ which can be permanent.
C. difficile colitis is widespread. The bacteria are passed in feces and spread to food, surfaces and objects when people who are infected do not wash their hands thoroughly. The bacteria produce hardy spores that can persist in a room for weeks or months. An estimated 500,000 cases are treated each year in North America and the problem is reported to be particularly severe in Canada and the UK. Annual death rates have been estimated at 20,000 for the US and 8,000 in the UK. A typical hospital stay for C. difficile colitis lasts from seven to ten days, so the costs of hospitalization alone are estimated at more than a billion dollars annually.
People in good health do not usually become ill from C. difficile. The intestines contain millions of bacteria, many of which naturally help protect the body from C. difficile infection. Antibiotics, taken to treat an infection, can destroy the normal, natural flora in addition to destroying the bacteria causing the illness. When the normal intestinal flora is compromised, C. difficile can quickly grow out of control. The antibiotics that most often lead to C. difficile infections include fluoroquinolones, cephalosporins, clindamycin and penicillins.
C. difficile disease is believed to be caused by the actions of two exotoxins, toxin A and toxin B, on gut epithelium. Both toxins are high molecular weight proteins (280-300 kDa) that catalyze covalent modification of Rho proteins, small GTP-binding proteins involved in actin polymerization, in host cells. Modification of Rho proteins by the toxins inactivates them, leading to depolymerization of actin filaments and cell death. Both toxins are lethal to mice when injected parenterally (Kelly and Lamont, Annu. Rev. Med., 49:375-90, 1998).
Exotoxins A and B which are produced by pathogenic strains of the bacterium are cytotoxic, enterotoxic, proinflammatory, and are considered to be the main virulence factors of this non-invasive microorganism. However, not all infections with toxigenic strains result in disease, prompting the search for additional virulence factors. Bacterial surface-expressed antigens represent candidate virulence factors, and are also considered important since such proteins likely mediate the essential functions such as adhesion to the epithelial layer of the gut in the first step of colonization or interaction with mediators of local immunity. In common with many other bacteria, C. difficile expresses a crystalline or paracrystalline surface layer (S-layer) on the outer cell surface. Such S-layers comprise proteins or glycoproteins forming a regularly arranged lattice on the external surface of the bacterium, and have previously been shown to be essential for the virulence of pathogens such as Aeromanas salmanicida and Campylobacter fetus. In contrast to most bacteria which comprise one S-layer, C. difficile is known to comprise two superimposed paracrystalline S-layers, each composed of a glycoprotein subunit which varies slightly in apparent molecular weight among different C. difficile strains. Most strains of C. difficile express two major S-layer proteins (SLPs), one of 32-38 kDa (low-MW SLP) and a second of 42-48 kDa (high-MW SLP). The low-MW SLP appears to be immunodominant and is the antigen most commonly recognized by patients suffering from C. difficile infections, and is the only antigen recognized in EDTA extracts of bacteria by antisera raised in rabbits against whole C. difficile cells (Calabi, E. et al., 2001, Mol. Microbiol., 40 (5) p 1187-99, PMID: 11401722).
The typical case of C. difficile colitis has one or more of the following characteristics: the patient is elderly and either lives in an institution or has been hospitalized for more than a week; the patient has undergone a course of antibiotics for an unrelated illness, often an upper respiratory infection or to prevent an infection after dental surgery; the patient has had C. difficile colitis before (the recurrence rate after successful therapy is roughly 20%).
C. difficile illness usually develops during or shortly after a course of antibiotics. Signs and symptoms may not appear for weeks or even months afterward. The most common symptoms of mild to moderate C. difficile disease are watery diarrhea three or more times a day for two or more days and mild abdominal cramping and tenderness.
In more severe cases, C. difficile causes the colon to become inflamed (colitis) or to form patches of raw tissue that can bleed or produce pus (pseudomembranous colitis). Signs and symptoms of severe infection include: Watery diarrhea 10 to 15 times a day, abdominal cramping and pain, which may be severe; fever; blood or pus in the stool; nausea; dehydration; loss of appetite; and weight loss.
As with many anaerobes, C. difficile forms spores that can accumulate on surfaces in institutions and may become airborne. These spores are resistant to many of the disinfectants typically used in hospitals and institutions. An aggressive strain of C. difficile has emerged that produces far more deadly toxins than other strains do. The new strain is more resistant to certain medications and has been found in patients who have not been in the hospital or taken antibiotics. This strain of C. difficile has caused several outbreaks of illness since 2000. Conventional therapy for C. difficile colitis includes administration of antibiotics such as bacitracin, cefprozil, meropenem, metronidazole, nitazoxanide, ticarcillin; clavulanic acid, tinidazole, and vancomycin. These antibiotics have severe side effects and are prescribed only when absolutely needed in order to delay the evolution of vancomycin-resistant strains of Clostridium and other bacteria.
If left untreated, C. difficile can lead to serious illness. Complications include dehydration, kidney failure, bowel perforation, toxic megacolon, and in some cases death. Severe diarrhea can lead to a significant loss of fluids and electrolytes, making it difficult for the body to function normally and possibly resulting in a drop in blood pressure. In some cases, dehydration can occur so quickly that kidney function deteriorates. C. difficile can cause extensive damage to the lining of the large intestine resulting in a perforated bowel. A perforated bowel can spill bacteria from the intestine into the abdominal cavity, leading to life-threatening peritonitis. The colon can become grossly distended when it is unable to expel gas and stool resulting in a toxic megacolon. Even in mild to moderate C. difficile infections, the infection can quickly progress to a fatal disease if not treated promptly.
In addition to antibiotic treatments, other therapies are reported to have a positive effect on the disease. For example, probiotics, the introduction of beneficial strains of bacteria through supplements and foods, have been shown to be beneficial in preventing C. difficile infections in populations of elderly and healthy people undergoing antibiotic therapies. Probiotics are organisms, such as bacteria and yeast, which help restore a healthy balance to the intestinal tract. A natural yeast called Saccharomyces boulardii, in conjunction with antibiotics, has been accepted as effective in helping prevent recurrent C. difficile infections. Probiotic therapy, however, does not directly inhibit the proliferation of C. difficile; rather, the therapy indirectly addresses the pathogen through competitive inhibition, that is, by promoting the growth of bacteria that compete with it in the confines of the intestines. Furthermore, the clinical literature does not support the use of probiotics alone in the treating C. difficile infections. In severe infections, surgery to remove a diseased portion of the colon may be performed.
Approximately 20% of people who have been infected with C. difficile get sick again, either because the initial infection never went away or because they become re-infected with a different strain of the bacterium. Treatment for recurrent disease may include: antibiotics, which may involve one or more courses of a medication, a longer course of treatment, or an antibiotic given once every two days; probiotics, such as S. boulardii, Lactobacillus acidophilus, Bifidobacterium bifidum, Bifidobacterium longum, and Bifidobacterium lactis given along with the antibiotic medication; or stool transplant (fecal bacteriotherapy) to restore healthy intestinal bacteria by placing donor stool in your colon. Although this is rarely done in practice, research has shown stool transplant to be helpful in selected cases.
In some implementations probiotics are microbial-based dietary adjuvants that beneficially affect the host physiology by modulating mucosal and systemic immunity as well as improving nutritional & microbial balance in the intestinal tract. [Naidu, N., Bidlack, W. R., Clemens, R. A. Probiotic Spectrum of Lactic Acid Bacteria (LAB), CRC Critical Reviews in Food Science and Nutrition, 39: 113-126, 1999.] In other implementations probiotics are used in a preparation of or a product containing viable, defined microorganisms with or without other substances in sufficient numbers, which improve or alter the micro flora or their properties (by implantation or colonization) in a compartment of the host and by that exert beneficial health effects in this host. Thus, probiotics may be applied to tissue extracts that stimulate microbial growth. It can also be deemed to mean organisms and substances which contribute to intestinal microbial balance or, alternatively, a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance.
Evidence indicates significant, positive health effects through the use of probiotics including, but not limited to: 1) reduction of Helicobacter pylori infection; 2) reduction of allergic symptoms; 3) relief from constipation; 4) relief from irritable bowel syndrome; 5) beneficial effects on mineral metabolism, particularly bone density and stability; 6) cancer prevention; and 7) possible reduction of cholesterol and triacylglycerol plasma concentrations.
Probiotic dietary supplements generally include organisms that are non-pathogenic and non-toxigenic, retain viability during storage, and survive passage through the stomach and the small intestine. Modification of the structure and metabolic activity of microflora is achieved through diet, primarily by administering probiotic live microbial food supplements. Different microorganisms prefer different habitats that may differ from host to host species. The best-known probiotics are the lactic acid-producing bacteria (i.e., Lactobacilli) and Bifidobacteria. Lactobacilli are a helpful type of bacteria naturally occurring in the intestines and constitute a major part of intestinal flora. Since all probiotics do not permanently colonize the host, they need to be ingested regularly for any health-promoting properties to persist. Commercial probiotic preparations generally comprise mixtures of Lactobacilli and Bifidobacteria. Different strains of probiotic bacteria may exert different effects based on specific capabilities and enzymatic activities, even within one species. Bacteria colonizing high-transit-rate sites, such as the small intestines, must adhere firmly to the mucosal epithelium and must adapt to the milieu of this adhesion site. The competition for adhesion receptors between probiotic and pathogenic microorganisms, therefore, is dependent on the habitat specifics.
Most lactic acid-producing or probiotic bacteria are extremely sensitive to common antibiotic compounds. Even during a normal course of individual antibiotic therapy, many individuals develop a number of deleterious physiological side-effects including: diarrhea, intestinal cramping, and sometimes constipation. These side effects are primarily due to the non-selective action of antibiotics as discussed herein. Thus, individuals taking antibiotics often suffer from gastrointestinal problems as a result of the beneficial microorganisms (i.e., intestinal flora), which normally colonize the gastrointestinal tract, being killed or severely attenuated. The resulting change in the composition of the intestinal flora can result in vitamin deficiencies when the vitamin-producing intestinal bacteria are killed, diarrhea and dehydration and, more seriously, illness should a pathogenic organism overgrow and replace the remaining beneficial gastrointestinal bacteria. Further, as a result of rapid evacuation of the bowels during diarrhea, a significant amount of the therapeutic compounds also do not get absorbed and are lost in the feces.
The interest in the use of novel, therapeutic agents other than antibiotics to treat C. difficile has grown over the past decades as the prevalence of infection has increased. Certain fatty acid esters have been noted for their potent antimicrobial effects. U.S. Patent Application No. 61/252,269 by Calvert et al., which is hereby fully incorporated by reference, discloses a novel liquid crystal delivery system comprised of antimicrobial compounds. Those compositions allow the use of medium chain glycerol fatty acids and/or their esters with enhanced solubility that provides broad range antimicrobial properties. Those antimicrobial compositions have been found to be extremely effective in inhibiting the growth of C. difficile. The most potent medium chain fatty acid has been found to be lauric acid (C12) and/or its monoglyceride glycerol monolaurate. The glycerol ester of lauric acid is a natural component of breast milk. Whereas infant nurseries commonly test positive for C. difficile, infant very rarely express the symptoms of C. difficile infection. A combination of a concentrated, soluble monolaurin with appropriate probiotic regimen administered at alternate intervals is a viable treatment for C. difficile infection while having no apparent negative effect on healthy or symbiotic Gram-positive or Gram-negative intestinal bacteria.
Even though glycerol monolaurate has been shown to be an effective broad range antimicrobial agent, low solubility and the formation of microcrystalline structures in situ have limited its use in practical applications. Glycerol monolaurate is typically used in concentrations in commercial formulations between 1-2%. Even at such low concentrations, formulations containing glycerol monolaurate are unstable such that the use of surfactants, emulsifiers, or other stabilizing agents is required.
Attempts to increase the solubility of glycerol monolaurate, and other fatty acids esters, diglycerides, triglycerides, etc. have been the focus of much research and development. It was found by U.S. Patent Application No. 61/252,269 by Calvert et al. that many of the common emulsifying mechanisms, for example the use of surfactants and emulsifiers with various hydrophile/lipophile balance values and combinations thereof, can render the active ingredient ineffective. That is, the act of emulsifying glycerol monolaurate with traditional emulsifiers reduces or eliminates its antimicrobial properties. Thus, the prior art has encountered a long-standing problem when attempting to include such anti-microbial agents as glycerol monolaurate at substantial concentrations while maintaining its anti-microbial effectiveness. Calvert et al. address that long-standing problem by providing formulations of highly soluble, stable liquid crystal mixture of biologically active fatty acid esters (salts and/or glycerol(s)) in an anhydrous polyhydric alcohol system in which the anti-microbial action of the fatty acid esters is maintained.
Monoglycerides are generally recognized as safe (GRAS) benign, non-toxic substances that are often used as emulsifiers for food and cosmetic products. Certain monoglycerides have been known to have powerful antimicrobial properties. Breast milk contains high amount of glycerides and it is suggested that infants employ these glycerides to protect against pathogens until their immune systems are fully functioning, some months after birth. Infant formulas typically contain coconut oil or other forms of lauric acid to provide similar protection. It has been well-documented that a paradox exists with regards to C. difficile that infants up to one year old have a much higher rate of asymptomatic carriage of C. difficile than all other populations, however they rarely develop C. difficile infections. Up to 50% of infants typically have detectable C. difficile as compared to approximately 3% of adults, but it is rare for infants to express colitis due to C. difficile. 
Polyglycerol and monolaurin are effective at inhibiting the growth of multiple genera of bacteria and parasites that reside in the intestines of a mammal which have the capacity of cause infection presented as diarrhea or colitis. Those species inhibited by polyglycerol and monolaurin include, but are not limited to C. difficile, Salmonella typhosa, Salmonella paratyphi, Salmonella schottmuelleri, Shigella dysenteriae, Shigella flexneri, Proteus vulgaris, Pseudomonas aeruginosa, Listeria, and Escherichia coli. In addition, Bacteroides species such as B. fragilis, B. uniformis, are also inhibited by polyglycerol and monolaurin.
The results of in vitro studies show that the stable liquid crystal mixture of biologically active fatty acid esters inhibits the growth of C. difficile at concentrations consistent with nutritional supplements.
There is thus a long-standing need in the medical community for highly effective compositions and methods for the treatment of bacterial infections, including those by C. difficile. By combining a highly soluble, stable liquid crystal mixture of biologically active, medium-chain fatty acid esters in a polyhydric alcohol solvent with broad-range antimicrobial efficacy with probiotic treatment, the present invention addresses that need.